There are a large number of different methods to count platelets in whole blood. Platelets can be counted under the phase microscope or light microscope. In addition, platelets can be counted by a variety of electronic instruments or particle counters. The two types of particle counters which are generally, commercially available are those which count particles by conductivity and those that employ light refraction to obtain the particle count. Typical commercially available counters include the "COULTER" ZBI Counter, the F.sub.n Counter and the Z.sub.f Counter from Coulter Electronics; the HPC-103 Counter from Hycel; and the TOA Platelet Counter PL-100. In addition, there is a light-dispersion type counter from Technicon Auto Counter.RTM. and the MK-4/HC.RTM. marketed by J. T. Baker.
Normal blood platelets range in size from 2.mu..sup.3 to 40.mu..sup.3 and in certain disease states they may be even larger. Consequently, for the automated counters to accurately count the number of platelets in the blood, they should count all platelets between 2 to 40.mu..sup.3 inclusive. Many of the commercially available instruments must be frequently calibrated to insure that only the proper size of particles are counted.
At present, manufacturers of these instruments set the instrument thresholds by using only a single standard of particles that have a known volume. For example, red blood cells have a volume of approximately 85.mu..sup.3. Quite often, these red blood cells are used to calibrate the instrument. Naturally, it can be seen that an instrument calibrated for red blood cells will not necessarily be calibrated for counting platelets. Other instrument users and manufacturers use a latex particle having a radius of 2.02 which provides a particle volume of approximately 4.3.mu..sup.3. This latter type of calibration principle using only one known point, assumes that the relationship between the settings and the volume is linear and the instruments are adjusted accordingly. However, this procedure of adjusting the instrument may take up to eight hours, depending upon the particular type of instrument being adjusted. Unfortunately, even after spending all this time, the instrument user or manufacturer still does not have an instrument that is accurately calibrated and which gives precise readings throughout the range for counting blood platelets.
Some of the commercially available instruments have thresholds that are preset at the factory and some are adjustable by the user. The problem of the improperly set instrument has become clear as the result of data obtained by the College of Pathologists and the report by Wertz and Koepke, "A Critical Analysis of Platelet Counting Methods", 68 American Journal of Clinical Pathology 195 (July 1977). Thus, it is well known that there is a problem to simply and quickly calibrate particle counting instruments which count extremely small particles having blood platelet sized volumes. However, the problem is perhaps more than just improperly set instruments and perhaps extends to an inability to determine if the instrument is calibrated or not. For example, in one of the many proficiency tests periodically taken and which was reported in the U.S. Department of Health, Education and Welfare, public health service report entitled, "Proficiency Testing Summary Analysis; Hematology 1977 II, Platelet Counting" (December 1977), the reported results revealed that the count of three reference samples expressed as number of platelets.times.10.sup.9 /1 varied from 234 to 750 for a sample having a reference mean count of 452 and a reference median count of 454, varied from 25 to 174 for a sample in which the reference count at a mean and a median of 76, and varied in a third sample from 76 to 442 for a sample in which the reference mean was 243 and the reference median was 232. Because the participating laboratories know that they were counting a test sample, it must be assumed that they used only calibrated instruments and that their results were thought to be accurate, however, the data received from the survey clearly indicates that the majority of the participating laboratories unknowingly had incorrect results which, in some cases, were off by a factor of 2.
Although particle counting systems are well known and commercially available, the problems typically present in these systems are discussed in the U.S. Pat. Nos. 3,392,331 to Coulter; 3,757,213 to Coulter et al; and 3,944,791 to Baxter; and in the references cited therein, all of which are incorporated herein by reference. On the other hand, U.S. Pat. Nos. to Gochman et al, 3,412,037; to Tate, 3,607,783; and to Butler, 3,791,192 disclose other problems of obtaining a calibration standard and are all incorporated herein by reference.
In summary, although platelet reference controls are a means for determining the reproducibility of pipeting and diluting procedures, they are not easily used to determine the appropriate settings for the counting instruments. This is because the reference control, like normal platelet-rich plasma, and like the conventional calibration standards, do not have an adequate concentration of particles at the upper and lower sizes.
It is noted that many of the commercial counting instruments are provided with means for adjusting the lower and upper threshold counts. It should be apparent, therefore, that the instruments must be adjusted to count all the particles within the known size distribution. If the instrument is adjusted to count particles having a size below the lower extreme size distribution, then other particles and perhaps electronic noise will be added to the true particle count. On the other hand, if the upper threshold level of the instrument is adjusted too high, an incorrect count may result from other particle impurities that may be present in the mixture being counted. Finally, if the upper and lower threshold settings are adjusted too low and too high, respectively, a threshold window that is too narrow will result and not all of the particles thought to be counted will actually be counted. Again, an incorrect and/or inaccurate count will be obtained.
It should also be apparent that any particle and carrier combination selected must be stable so as to avoid disintegration of the particles or the aggregation of two or more particles. If the particle size diminishes from disintegration or increases from aggregation, the particle may be too small or too large (i.e, fall outside the lower or upper threshold sizes) for the counting instrument to count. On the other hand, even if the resulting size of the smaller or larger particle does fall within the size capable of being counted by the instrument (i.e, within the threshold window), a different number of particles will be present in the standard than originally determined. Degradation of the particles can result not only from the instability of the substance from which the particle is made, but also from storage at improper environmental conditions and contamination from fungi and bacteria. The calibration standard can further be contaminated from the mechanical characteristics of the carrier in which the particles are suspended. An improper carrier may result in stratification of the particles or other types of uneven distribution of the particles in the carrier. Also, easy bubble formation would be an undesirable mechanical characteristic of the carrier. Finally, it should be obvious that the carrier must be stable over a wide range of environmental conditions and over a sufficient length of time.